Project Summary/Abstract Cilia have sparked phenomenal interest in the past decade, after the realization that they are a fundamental cellular organelle required for signaling. Untangling the specific mechanisms that regulate Sonic hedgehog (Shh) signaling within the cilium is difficult since so many mutants that disrupt ciliogenesis also affect Shh signaling. We have identified several mouse mutants disrupting cilia-related genes and Shh signaling using forward genetic screens aimed at identifying novel genes or new alleles of known genes that direct neural patterning. Our general strategy is to characterize the in vivo phenotype and then derive cell lines from the mutant mice to define cellular phenotypes. With knowledge gleaned from our colleagues in biochemistry and human genetics, we test mutant versions of the proteins, and then select some for in vivo modeling. Employing exactly this strategy, we have long focused on a small ciliary GTPase, Arl13b, that we hypothesize integrates the regulation of ciliogenesis and Hh signaling through distinct effectors and their downstream pathways. As a GTPase, single basepair mutations within the GTPase domain of Arl13b are predicted to disrupt individual effector pathways. Indeed, we have defined an Arl13b point mutation that disrupts the role of Arl13b in ciliogenesis but leaves the Shh response intact, indicating that the processes can be genetically uncoupled. In the next five years, using ARL13B mutants, a series of cell-based assays, and cell lines in which we can circumvent ciliogenesis or sensitize Hh disruption, we plan to unravel Arl13b function in ciliogenesis, cilia maintenance, traffic of proteins to/within cilia, and Shh signal transduction at unprecedented resolution. We expect Arl13b mutants will provide a genetic entry point from which we will identify at least a subset of effector proteins and define their mechanisms of action. The work in cell culture will enable us to select specific ARL13B mutants for which we can generate mouse models and bring our work full circle back to in vivo phenotypic characterization. In addition, we are integrating the novel alleles we discovered into our analysis. Thus, our proposal will generate a molecular genetic toolkit from which the field will be poised to distinguish the regulation of cilia from that of Hh signaling. This is important to our fundamental understanding of cilia, ciliogenesis, and cilia structure, as well as our basic comprehension of the Shh pathway.